The recent Ebola virus (EBOV) outbreak in West Africa, which took more than 10,000 lives in 2014, highlighted the critical need for robust treatment and prevention options to combat this highly virulent pathogen. One promising area of investigation uses non-infectious virus-like particles (VLPs) to mimic wild type viruses during vaccine construction. This strategy is the same one used to prepare human papillomavirus (HPV) vaccines.
Ongoing vaccine studies indicate that when administered with an adjuvant, EBOV VLPs (eVLPs) are protective against a lethal dose of EBOV in both mice and non-human primates. However, vaccine dosages require precise measurement of EBOV envelope glycoprotein subunit GP1 (N-terminal portion) concentration to ensure that each lot of eVLP vaccines meets quality standards for efficacy before release. To improve on the current assay methods (Western blotting or single-antibody ELISA), Cazares et al. (2016) recently turned to isotope dilution full-scan liquid chromatography–high-resolution mass spectrometry (LC-HRMS) for absolute quantitation of GP1 in eVLPs.1
The researchers selected two target peptides and synthesized isotopically labeled AQUA Ultimate peptides (Thermo Scientific) for each. The team noted their meticulous efforts to ensure complete tryptic digestion despite missed cleavages and the appearance of N-terminal truncated GP1 isoforms in the eVLP lots, highlighting the importance of peptide selection. After tryptic digestion and reverse phase purification, the researchers subjected dilutions of eVLP analyte digest and heavy AQUA standard to LC-HRMS using an an UltiMate 3000 high-performance LC system and an Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI-II) ion source (all Thermo Scientific).
Three replicates produced the following GP1 concentration data: 0.57 mg/ml, 0.49 mg/ml and 0.53 mg/ml, with an average of 0.53 mg/ml ± 0.04 mg/ml and a coefficient of variation of 7.6. This means that, on average, each aliquot of eVLP comprised 1.13 µg of GP1, or 11.3% of the 10 µg total protein concentration. The authors noted this data reflects the reproducibility of LC-HRMS for this purpose. They also reported a linear range for data over serial dilutions and quantitative accuracy to 1 fmol.
The researchers applied their optimized protocol to five separate eVLP lots (lots A through E) currently used for in-house animal vaccination studies. They reported a GP1 concentration range of 3.3 mg/ml to 7.2 mg/ml for LC-HRMS compared with 0.71 mg/ml to 1.4 mg/ml for Western blot. The team indicated that this difference likely resulted from the appearance of N-terminal truncated GP1 isoforms previously described as well as the presence of GP2 (the C-terminal portion of the envelope glycoprotein).
Finally, the research team immunized mice (n = 20) using all five eVLP lots and compared survival data after EBOV challenge. They found the lowest incidence of survival (40%) in animals receiving lot E vaccines and the highest incidence of survival (100%) for those that received lot A or lot B vaccines. This is significant, because lot A contained the highest GP1 concentration, as determined by LC-HRMS, while lot E contained the lowest GP1 concentration. Using the data acquired here, the researchers determined the actual concentration of antigenic GP1 was 2.89 µg per 10 ug GP1. They reported this as the minimal vaccination dose for 100% survival.
Overall, Cazares et al. highlight that the traditional method (Western blot) overestimated GP1 concentration in eVLP because it did not distinguish between GP1 and related proteins (i.e., GP2 or truncated GP1 isoforms). For this reason, only LC-HRMS data correlated with survival rates after EBOV challenge. The researchers indicate that this improved assay will enable more precise monitoring of eVLP batch variability, optimize vaccine production and aid in determining adequate dosing.
1. Cazares, L.H., et al. (2016) “Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations,” Clinical Proteomics, 13(1) (18), doi: 10.1186/s12014-016-9119-8.